A protocol for capturing RNA-sensing innate immune receptors in multiple conformations by single-particle cryo-EM

Summary Capturing different conformations of receptor proteins that are complexed with ligands by single-particle cryo-EM facilitates our understanding toward the mechanisms of ligand recognition and receptor activation cascades. Here, we present a protocol for capturing RNA-sensing innate immune receptors, such as RIG-I, in multiple conformations by single-particle cryo-EM. We describe steps for protein-ligand sample preparation, data acquisition, and image processing covering focused three-dimensional classification. This protocol can be adapted to capture the dynamic behavior of other receptors that can be stabilized. For complete details on the use and execution of this protocol, please refer to Wang and Pyle (2022).1

ii. Add 25-35 mL cultured seeds to 1 L LB media containing antibiotics at same concentration as described above. iii. Add IPTG (Final, 0.5 mM; Stock, 1 M in ddH 2 O) to induce RIG-I overexpression when OD600 reaches 0.6, and proceed for 20-24 h at 16 C and 180 RPM. b. Collect cell pellets by centrifugation at 6,000 3 g for 10 min, then put pellets to À80 C for storage.
Pause point: The pellets can be stored at À80 C for 1 month, but fresh bacteria is recommended for protein purification.
a. Weigh 10 g total bacteria pellets. i. Lyse them using a microfluidizer in the lysis buffer (10 mL/g) supplemented with EDTA-free Protease Inhibitor Cocktail. ii. Centrifuge cell lysate at 15,000 3 g for 30 min and collect the supernatant. b. Equilibrate 3 mL of 50% slurry Nickle beads (by volume) with over 30 mL ddH 2 O and 30 mL lysis buffer, respectively. c. Mix the supernatant with the equilibrated beads, and stir the mixture at low speed, which does not produce abundant bubbles, at 4 C for 1 h. d. Pour the slurry to a gravity column. Wash beads with 20 mL wash buffer 1, followed by 20 mL wash buffer 2. e. Elute RIG-I with 10 mL elution buffer. 3. Purify RIG-I by heparin affinity chromatography and size-exclusion chromatography. Troubleshooting 1. a. Mix 20 mL lysis buffer with the 10 mL elution. Add 0.5 mg ULP1 to cleave SUMO tag from 2.5 mg eluted protein (yield from 10 g bacteria) at 4 C for 1 h or 12-16 h. b. Add 20 mL low salt buffer to 30 mL RIG-I solution.
i. Then load the 50 mL solution onto a 5 mL HiTrap Heparin HP column at 2 mL/min at 4 C.
ii. Elute RIG-I at 1 mL/min using 65% high salt buffer. c. Concentrate the eluted RIG-I to 0.5 mL using a 50 kD Amicon Ultra-4 centrifugal filter unit.
i. Load RIG-I onto a Superdex 200 Increase 10/300 GL column with gel filtration buffer at 0.5 mL/min at 4 C. ii. Collect the peak fractions of monomeric RIG-I eluted around 13.3 mL ( Figure 1A). d. Pool RIG-I fractions in a storage buffer at concentration of 10-20 mM, flash freeze RIG-I in LN 2 and store RIG-I at À80 C for further experiments.
Pause point: The purified RIG-I can be stored at À80 C for at least one year.  c. Purify transcribed RNA by gel extraction from 12%-20% urea denaturing polyacrylamide gel and assess its purity by mass spectrometry (Novatia) (Figures 1B and 1C). d. Treat p3SLR30 with calf intestinal alkaline phosphatase (CIP) to remove the triphosphate group. Assess OHSLR30 purity by mass spectrometry (Novatia) ( Figure 1C). 6. Anneal RNA duplexes (p3dsRNA, p2dsRNA, p1dsRNA, OHdsRNA) and stem loop RNA (p3SLR30, OHSLR30). a. Anneal RNA duplexes (260 mM, 50 mL) by rapidly heating to 99 C and slowly cooling over 1 h to 4 C in the annealing buffer (200 mM NaCl) on a Thermocycler. b. Anneal stem loop RNA (60 mM) by heating to 90 C and incubate at 90 C for 2 min and then snap-cooled on ice for 30 min. c. Assess quality of annealed RNAs by running samples on a 15% native polyacrylamide gel (Figure 1D).

RNA preparation
Pause point: The ssRNAs and dsRNAs can be stored in ddH 2 O at À80 C for at least one years.

MATERIALS AND EQUIPMENT
Note: The pH is adjusted with 3 M NaOH. The buffer without BME is stored at 4 C for up to 3 months. BME is freshly added before usage.
Note: The pH is adjusted with 3 M NaOH. The buffer without BME and Imidazole is stored at 4 C for up to 3 months. BME and Imidazole are freshly added before usage.
Note: The buffer is freshly made with lysis buffer. BME and Imidazole are freshly added before usage. Note: The buffer is freshly made with lysis buffer. BME and Imidazole are freshly added before usage.
Note: The pH is adjusted with 3 M NaOH. The buffer without BME is stored at 4 C for up to 3 months. BME is freshly added before usage.
Note: The pH is adjusted with 3 M NaOH. The buffer without BME is stored at 4 C for up to 3 months. BME is freshly added before usage.
Note: The pH is adjusted with 3 M NaOH. The buffer without BME is stored at 4 C for up to 3 months. BME is freshly added before usage.
Note: The pH is adjusted with 12 M HCl. The buffer is stored at 4 C for up to 1 month.

STEP-BY-STEP METHOD DETAILS
Sample preparation for freezing grids

Timing: 16 h
These steps describe the purification of RNA complexes and preparation for freezing grids.
Note: It is well established that the footprint size of RIG-I on dsRNA is 8-12 bp. We found that lengthening the RNA stem (14 bp to 24 bp) had several technical advantages and it improved our ability to image the particles due to the inherently high contrast of RNA duplexes (Global resolution was improved from 3.8 Å to 3.5 Å ). Note: These concentrations are used because they achieve decent particle density on grid (Figure 2A). Note: These concentrations are used because they achieve good particle density on grids (Figure 2A). a. Assemble the dry cryogen container (liquid nitrogen reservoir, float, brass cryogen cup, metal storage box holder, metal ''spider legs'' and labeled grid boxes). b. Cool the container by filling with liquid nitrogen (LN 2 ) to the brass cryogen cup. c. After the container is cooled, fill the reservoir with LN 2 again, but do not add to the brass cup.
Note: After the container is cooled, make sure the brass cryogen cup does not contain LN 2 . Note: As liquid ethane in cryogen cup will solidify in a short time, it is recommended to fill with liquid ethane right before freezing grids. 9. Hold one grid with Vitrobot tweezer and mount the tweezer to Vitrobot.
a. Lift the tweezer with grid, then transfer and lift the container. b. Apply 3 mL samples to the carbon side of the grid, blot samples with force -4 for seconds (2, 3, 4, 5 s), followed by plunging grid to liquid ethane. c. Transfer the grid to one of the four slots of the grid box. Troubleshooting 3. 10. Repeat step 9 with adjusted blotting time to freeze more grids. Troubleshooting 4. 11. Clip the frozen grids using the C-clip and the C-clip ring in the grid clipping station (ThermoFisher), and store the autogrids (clipped grids) in the autogrid boxes.
Note: Sometimes solid ethane covers the grid surface. To prevent the solid ethane from damaging the grid during grid clipping, the grids can be left in LN 2 for 1-2 days to remove the solid ethane.
12. Transfer the autogrid boxes to the LN 2 storage dewar for grid screening and data collection.
Pause point: The grids can be stored in LN 2 dewar for months before loading to electron microscope for grid screening and data collection, but it is better to restrict it within 2 months.

Cryo-EM data acquisition
Timing: 1-3 days These steps describe the grid screening and cryo-EM data collection. 13. Mount the grids to the Glacios using the autoloader and screen the grids.
a. Take a full map of the grids using the software SerialEM 5 and check the ice thickness. b. Select grids with thin ice and pick 2-3 squares with ice of different thickness.
Note: The squares where most holes are visible but the square edges are not sharp usually have decent ice thickness for grid screening and data collection.
i. Select 2-3 holes in one square, take micrographs from either center or edge of the holes.
Alternatives: Both Glacios and Krios are suitable to collect high-quality data for reconstructing high-resolution cryo-EM maps.
a. Set up the data collection conditions, make sure the beam is ready for high-resolution data collection. Defocus range (mm) b. Collect the micrographs with calibrated pixel size of around 1 Å /pix and total electron exposure of 50-60 e -/Å 2 (total electron exposure/frame, 1-1.5 e -/Å 2 ). c. Use different defocus ranges during data acquisition (Table 1).

ll OPEN ACCESS
Alternatives: When the particles are still visible by eyes under low defocus range (low, -0.8$-2.0 mm; high -1.2$-3.0 mm), low defocus range can be applied to improve the resolution. d. For each stage movement, acquire one micrograph/hole of 9 holes on R1.2/1.3 grid.
Alternatives: For R2/2 grid, three to five micrographs/hole can be collected.
e. Collect about 3000 micrographs under super-resolution mode in one day. Generate the jpg picture during data collection for monitoring the image quality.
Alternatives: The required number of micrographs depends on number of autopicked particles required for data processing (1 million particles are a good start.).

Cryo-EM data processing
Timing: 2-3 weeks These steps describe the process of reconstructing high-resolution maps of different conformations (Table 1). 15. Process datasets through Relion. 8 The micrographs are dose-weighted and beam induced motion corrected through MotionCor2. 6 a. With 5 3 5 patches, bin twice with micrographs collected under super-resolution mode. b. Select micrographs with total motion below 40 Å for CTF estimation. 16. Estimate the CTF parameters using CTFFIND4 7 with the non-dose-weighted and motion corrected micrographs. a. Estimate the CTF of selected micrographs with FFT box size of 1024 pix. b. Select the micrographs with resolution above 6 Å . c. Manually evaluate the selected micrographs; Dispose of micrographs with bad CTF fitness of Thon rings and ice contamination. 17. Pick up particles from selected micrographs using autopick.
Note: Adjust the scale and sigma of micrographs to help pick up particles. Sometimes small scale and sigma help people easily figure out the single particles.
ii. Perform 2D classification with the manually picked particles using 10-20 classes.
iii. Select the 2D classes showing features of complex ( Figure 2B).
Alternatives: The previously resolved cryo-EM map of the sample can be used as the 3D reference of autopick. The pixel size and box size of the map should be adjusted to the same as the binned micrographs using relion_image_handler command.

OPEN ACCESS
ii. Manually assess the autopick results. Make sure most of the particles are picked up; Unpick the ice and carbon.
Alternatives: The Gautomatch (developed by Prof. Kai Zhang, https://www2.mrc-lmb.cam.ac. uk/download/gautomatch-056/) can also be used for autopicking if Relion autopick fails to pick up most of the particles possibly due to low defocus for data collection. 18. Perform 2D classification of the autopicked particles. a. Downscale the particles by a factor of two using Extract job to accelerate data processing and save computational resources. b. Perform 2D classification using 100 classes. c. Select classes with features of particles and dispose of classes representing misfolded samples or ice contamination ( Figure 2C).
Note: Only discard classes that are certainly contamination. The bad particles derived from seemingly bad 2D averages can be removed later during 3D classification in step 19. To improve the resolution, erase the unbound end of dsRNA in the 3D map generated above through Chimera, 10 and apply a soft mask (Lowpass filter map, 15 Å ; Initial binarization threshold, 0.002; Extend binary map this many pixels, 6; Add a soft-edge of this many pixels, 6) to the modified map to re-implement the 3D classification with selected particles from the 2D classes ( Figure 2D). c. With particles from the best 3D class, perform masked 3D refinement (Initial low-pass filter, 60 Å ; Use solvent-flattened FSCs, Yes), Ctfrefine (Minimum resolution for fits, 30 Å ; Perform CTF parameter fitting, Yes; Fit per-particle defocus, Yes; Range for defocus fit, 2,000 Å ; Perform beamtilt estimation, Yes) and Byaesian polishing (Optimized parameters using 10,000 particles, then use the optimized parameters to polish micrographs), followed by a final 3D refinement. 8,14 Note: For samples containing multiple conformations, particles from the best 3D class could be applied to another 3D classification (5-10 classes, T = 4) without alignment, thereby classifying particles into 3D classes of different conformations. Then these maps of different conformations can be subjected to subsequent 3D refinement etc ( Figure 3B).
d. Perform postprocessing job to yield maps at near-atomic global resolution ranging from 2.9 Å to 3.7 Å (Table 1) according to the FSC = 0.143 criterion and sharpened with B-factor. 15

EXPECTED OUTCOMES
In this step-by-step protocol, we described the detailed steps of sample preparation, grid freezing, data acquisition and data processing. Following this protocol, we have successfully reconstructed the cryo-EM maps of RIG-I in different conformations in the presence of different dsRNAs and ATP. 1 This protocol can be further utilized to explore structures of RIG-I:RNA complexes from heterogeneous samples, and adapted to study other similar receptors.

QUANTIFICATION AND STATISTICAL ANALYSIS
All the datasets were processed using Relion, assisted with Chimera.

LIMITATIONS
Although this protocol was successfully used to explore different RIG-I conformations, it may not be utilized to study other macromolecular complexes without adaptation, since each complex shows distinctive characteristics. To capture the sub-state conformations, it's more worthwhile stabilizing the complex in these conformations by biochemistry methods, such as assembly with other or more binding partners, introduction of truncations and mutations, addition of compounds and change of buffer conditions. When these sub-state conformations stably exist in solution, it is expected to be feasible to use the cryo-EM technique in this protocol to resolve these structures, otherwise, the time-resolved cryo-EM technique might be a better option capturing the transient unstable sub-state conformations.

Problem 1
Fail to obtain monomeric RIG-I protein.
Refer to steps 1 and 3 of protein preparation section.

Potential solution
Inducing RIG-I expression at high OD600 might lead to more RIG-I aggregates. It is good practice to reduce RIG-I aggregates by inducing RIG-I expression when OD600 achieves 0.6.
The His tagged SUMO tag might be removed before recovering samples with Ni Beads. This can be resolved by using fresh bacteria or short-term (one month) stored bacteria at À80 C.

Problem 2
There are few particles per micrograph. Refer to step 7 of cryo-EM grid preparation.

Potential solution
To trap more particles in the hole, it is worth trying to increase sample concentration, testing different grids, such as grids of different brands (quantifoil, c-flat), grids with different support (Cu, Au), grids without carbon (UltraAu), or grids coated with a single carbon or graphene oxide layer. Reverse blotting (glow discharging on one side and applying samples to the other side) might help as well.

Problem 3
Ice contamination including crystalline ice. Refer to step 9 of cryo-EM grid preparation.

Potential solution
Make sure the cryogen container is cooled thoroughly. Be careful when transferring frozen grids from liquid ethane to LN 2 and from Autogrid box to autoloader. The transient exposure to ambient temperature might cause thaw-freeze cycle which usually leads to crystalline ice. Use clean fresh LN 2 can reduce chunk of ice as well.

Problem 4
Ice is either too thin or too thick. Refer to step 10 of cryo-EM grid preparation.

Potential solution
It is hard to reproduce the grid even under same freezing conditions. To freeze grids with optimal ice thickness, it is recommended to freeze a series of grids using different blotting time on the same batch.

Problem 5
Poor data quality of micrographs. Refer to step 14 of cryo-EM data acquisition.

Potential solution
During data acquisition, keep an eye on the collected micrographs. If the micrographs show abnormal features, such as small file size and incomplete view, stop data collection and check if the microscope works as expected.

Problem 6
Only poor 3D class maps are obtained after 3D classification. Refer to step 19 of cryo-EM data processing.

Potential solution
To obtain a good 3D map from a dataset for the first time, sometimes this requires multiple rounds of 3D classification (10 rounds). After the 3D map with features of secondary structures shows up, this map can be used as 3D reference of 3D classification with selected particles from 2D classification. This time it only requires one or two rounds of 3D classification.
If there is a reliable high-quality cryo-EM map of samples similar to the complex, this map can be used as 3D reference of 3D classification.
If all these above do not work, review the autopick and 2D classification jobs, and check whether most of the particles are picked up and whether 2D classes show clear features of secondary structures.
If the particles are picked up properly but 2D classes don't have distinctive features, the sample should be further optimized.

RESOURCE AVAILABILITY
Lead contact All requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Anna Marie Pyle (anna.pyle@yale.edu).

Materials availability
All materials will be available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability
Atomic coordinates and cryo-EM maps are deposited in EMDB and PDB as described in key resources table and the published paper. 1 This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.